The focus of the experiments described in this research proposal Is the identification and characterization of the erythroid specific cis- elements of the mouse band 3 gene Band 3 is the predominant transmembrane polypeptide of the mature mammalian erythrocyte where it functions as an anion exchanger and also serves as the critical attachment point of the spectrin-based membrane skeleton. Band 3 plays a pivotal role in the assembly of the membrane skeleton during red cell differentiation. In early CFUe cells which do not yet synthesize band 3, newly synthesized ankyrin, spectrin, and 4.1 polypeptides are assembled only transiently into a skeleton that is unstable; the assembly of these components into a stable membrane skeleton coincides with the onset of band 3 protein synthesis at the early normoblast stage of maturation. Several observations summarized in Background and Significance suggests that band 3 protein plays pivotal roles in regulating the stoichiometric assembly of the individual components of the membrane skeleton into a stable structure and in localizing its assembly to the underside of the plasma membrane. The initiation of band 3 gene expression during erythropoiesis, therefore, may be a key step in the progression of red cell progenitors into a terminal differentiation pathway. Although a great deal is known about the structure and function of band 3 protein in immature and mature red cells, very little is known about the regulation of band 3 gene expression. Our preliminary experiments and work from other laboratories indicate that the mouse band 3 gene has upstream and internal promoter cons. The principal erythroid transcript is derived from an upstream promoter and encodes a full-length transcript and protein, whereas the principal kidney transcript is derived from a separate internal promoter and encodes a 5'-truncated transcript and N-terminal truncated protein. We propose to study the tissue specificities of the upstream and the internal band 3 gene promoters more thoroughly using in situ hybridization techniques to detect full-length and truncated band 3 mRNA transcripts in various erythroid and nonerythroid tissues of adult mice. This approach will be complemented using in situ immunological techniques to detect full-length and truncated band 3 proteins. The mouse band 3 gene upstream regulatory region (URR) contains a promoter element that is used primarily in erythroid cells. We will identify and characterize the we-elements of the mouse band 3 gene upstream regulatory region (URR). Using transient expression techniques, we will identify positive and negative regions of the URR. We will attempt to identify erythroid- specific cis-elements that are responsible for the activation of band 3 gene transcription. We will approach this question using transient expression experiments and DNAse I and DNA gel shift assays to study control and induced red cell nuclear extracts. The overall goals of this proposal are to identify and characterize the promoter elements of the mouse band 3 gene that regulate its expression during erythropoiesis. The long term goals of this project are to understand how band 3 gene expression is restated during erythropoiesis in coordination with other erythroid genes, and to define more completely, using molecular and cellular approaches, the roles played by band 3 protein in erythropoiesis. It is expected that information obtained from this work will also be useful in our future studies of patients with red cell defects characterized by band 3 protein deficiency.